This doctoral thesis was dedicated to the study of the secondary transporter, the melibiose permase (MelB) from Escherichia coli with biochemical and biophysical methodologies. The main objective was to obtain insights of the symport mechanism of MelB. This prokaryotic transporter uses the downhill translocation of a cation to transport the disaccharide melibiose against its concentration gradient into the cell. Although MelB possess the highest affinity for Na+ cations, the transporter can couple the transport process also to the smaller Li+ and H+ ions. Apart from melibiose, MelB transports a variety of α- and β-galactosides making it versatile carrier. The transporter comprise 70% of hydrophobic amino acids and is organized in 12 transmembrane spanning helices connected by hydrophilic loops. As preliminary studies already reveal, the C-terminal domains of MelB has been proven to play a crucial role in the active transport mechanism. The focus in this doctoral thesis lies especially on the helices X and XI as well as the interconnecting cytoplasmic loop 10-11. Site-directed mutagenesis delivered valuable information about important amino acids which might participate in the active transport by forming part of the binding sites or taking part in the translocation and release of the substrates. During our study, numerous MelB mutants have been extracted from the membrane by using the detergents LAPAO and β-DDM. Subsequently, the solubilized transporter was reconstituted into liposomes composed of lipids from E. coli mimicking MelB's original habitat. As the only charged residue residing in the transmembrane segment XI, Lys-377 was replaced by cysteine, valine, arginine, histidine and aspartic acid. None of these MelB mutants interacts with sodium concluded from infrared difference spectra (IRdiff) induced by the cation Na+. The melibiose binding is only remotely detectable in K377R, K377H and K377C in the presence of the proton. K377V and K377D lost the interaction with the sugar molecule. The charge in the particular position in MelB is important but not sufficient to conserve the substrate binding. The amino acid substitutions of Lys-377 appear as white colonies on a MacConkey agar plate. This color indicates the absence of hydrolysis of the melibiose into the monosaccharides, glucose and galactose. A metabolisation of the sugar is indicated by a red colony. In these colored bacteria, the sugar hydrolysis causes an acidification of the medium indicated by the MacConkey agar which stains the bacterial colonies. The screening of potential revertants, second site mutations which allow sugar influx, demonstrate the appearance of a unique second site mutation I22S which turn the former white colonies of the Lys-377 mutants into a red phenotype. Another red-colored phenotype appeared only for the mutant K377R, in which Leu-236 is replaced by a phenylalanine. All K377/I22S revertants are not able to restore the melibiose binding in proteoliposomes and only the revertant K377C/I22S recovered a partial sodium binding. The absorbance spectra of the single Lys-mutants as well as the corresponding revertants indicate a correlation between the absence of sodium binding and conformational changes of the MelB transporter. These structural changes are identical to previously examined MelB mutants D55C, D59C and D124C which also demonstrated a detrimental impact on the Na+ binding. The single mutants I22S and I22A exhibit a clear response of the structure in the presence of Na+. The interaction with melibiose on the contrary is lost in these MelB mutants. This outlines Ile-22 as a potential amino acid participating in the binding process of the melibiose molecule in MelB. The second revertant K377R/L236F demonstrates only minor interaction with the Na+ as well with the melibiose in proteoliposomes. Measurements using membrane vesicles of this mutant demonstrate however a large Förster energy transfer (FRET) signal mediated by the fluorescent sugar analog, D2G. By extracting the mutant K377R/L236F with β-DDM instead of LAPAO, the solubilised and then reconstituted transporter exhibits a clear improvement considering the conservation of the MelB structure. IRdiff spectroscopy results concluded that the K377R/L236F mutant binds melibiose and Na+, although the cation-induced difference spectra differs from the C-less reference. The PCR-generated double mutant K377C/L236F failed to demonstrate similar interactions with the substrates in proteoliposomes as well as in membrane vesicles. The positive charge in position 377 is essential for the MelB transporter, but the sole charge is not sufficient for the substrate binding. The charge requires also structural arrangement to interact with the sugar and the co-ion. In the second part of the PhD thesis, cysteine mutants replacing charged residue in the cytoplasmic loop 10-11 were characterized for their ability of substrate binding. The transport-defective mutants D351C, D354C and R363C couple sodium with reduced affinity indicated by their low intense IRdiff spectra. The melibiose binding is even further reduced in these MelB mutants. Appearing as white colonies on MacConkey agar plate, the screening of potential revertants only revealed a second site mutation for D354C. Interestingly, Ile-22 was substituted for a serine like in the Lys-377 mutants. Congruent results with the K377/I22S mutants indicate for D354C/I22S the loss of sugar binding. However, the sodium triggers a difference spectrum similar to C-less and much more intense than in the D354C single mutant. This indicates a tight interaction of the cation with the MelB mutant D354C/I22S. In the third part of this thesis, eight mutants were generated by replacing crucial residues in the C-terminal helix XI of MelB with cysteine. The mutants Q372C, G379C, F385C, L391C and G395C show an almost normal interaction with Na+. A383C and Y396C on the other side display a Na+-induced IRdiff with low intensity indicating their impaired cation binding. Considering the interaction with melibiose, all helix XI mutants bind the sugar in the presence of either the proton or Na+. The MelB mutants A383C, L391C and Y396C exhibit a melibiose-induced difference spectrum with very low intensity indicating their importance for the binding process. Tested in vesicles and proteoliposomes, G379V is the only characterised mutant which lacks sodium and sugar binding making this mutant a potential candidate for crystallography trials to obtain the structure of the empty MelB transporter.